Treatment of HIV-1 infection with highly active antiretroviral therapy (HAART) in the past decades has remarkably reduced HIV/AIDS related mortality and morbidity. However, the emergence of drug resistance in persons on antiretroviral therapy (ART) and the transmission of drug-resistant HIV strains to newly infected persons are a major threat to the global effort for HIV prevention and treatment success [1, 2, 3]. Recently, access to antiretroviral drugs (ARVs) has been scaled up rapidly in resource-limited countries where availability of laboratory monitoring is often limited or lacking [4, 5]. This creates the potential for HIVDR emergence and transmission in these settings. Detection and monitoring of HIVDR by molecular genotyping is pivotal to ensure ongoing regimen efficacy. It is the standard of care in resource-rich countries [2, 6]; however in resource poor countries, HIVDR testing is not generally available or it is too costly to be used in routine monitoring of patients receiving ARVs. Therefore, the World Health Organization (WHO) recommends population-based surveillance and monitoring of drug resistance (DR) in resource-limited settings [2, 4, 7]. Pattern and rates of transmitted and acquired drug resistant HIV variants will collectively inform regional and global recommendations on which ARVs to maintain or change in treatment regimens [7].
Population sequencing-based genotyping methods including ViroSeq, TRUGENE and in-house assays are widely used and the most informative and affordable genotyping methods for monitoring patients on ART in clinical practice [8, 9, 10, 11]. However, ViroSeq and TRUGENE, the two FDA-approved genotyping assays were designed for HIV-1 subtype B viruses which are the predominant HIV-1 strains in resource-rich countries. In addition, these commercial kits are expensive and less sensitive to non-B subtypes, limiting the application in resource-limited settings [12, 13, 14]. There have been no commercially available HIV-1 genotyping assays designed for non-B subtypes and CRFs that are predominant viral strains in resource-limited countries. Moreover, the demand for low cost and sensitive genotyping methods is increasing with the establishment and expansion of laboratory molecular monitoring in these settings [15, 16].
The most frequently used HIVDR genotypic assays are assays that detect resistance mutations in the reverse-transcriptase (RT) and protease (PR) genes [17, 18, 19]. The minimal genotyping requirements for these two regions are PR codons 10-99 and RT codons 41-240 [3, 20, 21]. An original in-house assay [22] had limitations: (1) it does not cover the entire PR gene region required for resistance testing; and (2) for some HIV-1 subtypes or CRFs, some sequencing primers generate higher background noise which affected the detection of mixture bases. Thus, a new assay is needed.
In-house assays are relatively inexpensive and sensitive for multiple subtypes, but in-house assays should only be implemented after adequate validation, including evaluating assay's performance with various HIV-1 subtypes [3, 20, 23]. Factors that could contribute to genotyping quality include type of assay/kit used, specimen handling and storage, level of experience of technicians performing the analysis, heterozygosity of sequences, and viral subtypes in clinical samples [11, 21].